1. Field of Invention
This invention relates to salt forms of 6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one, which is a selective cyclin-dependent kinase 4 (CDK4) inhibitor that is useful for treating inflammation and cell proliferative diseases such as cancer and restenosis.
2. Discussion
Cyclin-dependent kinases and related serine/threonine protein kinases are important cellular enzymes that perform essential functions in regulating cell division and proliferation. The cyclin-dependent kinase catalytic units are activated by regulatory subunits known as cyclins. At least sixteen mammalian cyclins have been identified (D. G. Johnson and C. L. Walker, Annu. Rev. Pharmacol. Toxicol. (1999) 39:295-312). Cyclin B/CDK1, Cyclin A/CDK2, Cyclin E/CDK2, Cyclin D/CDK4, Cyclin D/CDK6, and probably other heterodimers including CDK3 and CDK7 are important regulators of cell cycle progression. Additional functions of Cyclin/CDK heterodimers include regulation of transcription, DNA repair, differentiation and apoptosis (D. O. Morgan, Annu. Rev. Cell. Dev. Biol. (1997) 13261-13291).
Cyclin-dependent kinase inhibitors may prove useful in treating cancer. Increased activity or temporally abnormal activation of cyclin-dependent kinases has been shown to result in the development of human tumors (C. J. Sherr, Science (1996) 274:1672-1677). Indeed, human tumor development is commonly associated with alterations in either the CDK proteins themselves or their regulators (C. Cordon-Cardo, Am. J. Pathol. (1995) 147:545-560; J. E. Karp and S. Broder, Nat. Med. (1995) 1:309-320; M. Hall et al., Adv. Cancer Res. (1996) 68:67-108). Naturally occurring protein inhibitors of CDKs such as p16 and p27 cause in vitro growth inhibition in lung cancer cell lines (A. Kamb, Curr. Top. Microbiol. Immunol. (1998) 227:139-148).
Small molecule CDK inhibitors may also be used in the treatment of cardiovascular disorders such as restenosis and atherosclerosis and other vascular disorders that are due to aberrant cell proliferation. Vascular smooth muscle proliferation and intimal hyperplasia following balloon angioplasty are inhibited by over-expression of the cyclin-dependent kinase inhibitor protein p21 (M. W. Chang et al., J. Clin. Invest. (1995) 96:2260; Z-Y. Yang et al., Proc. Natl. Acad. Sci. (USA) (1996) 93:9905). Moreover, the purine CDK2 inhibitor CVT-313 (Ki=95 nM) resulted in greater than 80% inhibition of neointima formation in rats (E. E. Brooks et al., J. Biol. Chem. (1997) 29207-29211).
CDK inhibitors can be used to treat diseases caused by a variety of infectious agents, including fungi, protozoan parasites such as Plasmodium falciparum, and DNA and RNA viruses. For example, cyclin-dependent kinases are required for viral replication following infection by herpes simplex virus (HSV) (L. M. Schang et al., J. Virol. (1998) 72:5626) and CDK homologs are known to play essential roles in yeast.
Selective CDK inhibitors can be used to ameliorate the effects of various autoimmune disorders. Rheumatoid arthritis, a chronic inflammatory disease, is characterized by synovial tissue hyperplasia. Inhibition of synovial tissue proliferation should minimize inflammation and prevent joint destruction. Expression of the CDK inhibitor protein p16 in synovial fibroblasts has been found to inhibit growth (K. Taniguchi et al., Nat. Med. (1999) 5:760-767). Similarly, in a rat model of arthritis, joint swelling was substantially inhibited by treatment with a p16 expressing adenovirus. CDK inhibitors may be effective against other disorders of cell proliferation including psoriasis (characterized by keratinocyte hyperproliferation), glomerulonephritis, and lupus.
Certain CDK inhibitors may be useful as chemoprotective agents through their ability to inhibit cell cycle progression of normal untransformed cells (Chen et al. J. Natl. Cancer Institute (2000) 92:1999-2008). Pre-treatment of a cancer patient with a CDK inhibitor prior to the use of cytotoxic agents can reduce the side effects commonly associated with chemotherapy. Normal proliferating tissues are protected from the cytotoxic effects by the action of the selective CDK inhibitor.
Review articles on small molecule inhibitors of cyclin-dependent kinases have noted the difficulty of identifying compounds that inhibit specific CDK proteins without inhibiting other enzymes. Thus, despite their potential to treat a variety of diseases, no CDK inhibitors are currently approved for commercial use (P. M. Fischer, Curr. Opin. Drug Discovery (2001) 4:623-634; D. W. Fry and M. D. Garrett, Curr. Opin. Oncologic, Endocrine & Metabolic Invest. (2000) 2:40-59; K. R. Webster and D. Kimball, Emerging Drugs (2000) 5:45-59; T. M. Sielecki et al., J. Med. Chem. (2000) 43:1-18.).
Despite these difficulties, recent studies have identified a number of selective CDK4 inhibitors that, as discussed above, may prove useful in treating cancer—either as anti-cancer agents or as chemoprotective agents—and in treating cardiovascular disorders, such as restenosis and atherosclerosis, diseases caused by infectious agents, and autoimmune disorders, including rheumatoid arthritis. For a disclosure of these selective CDK4 inhibitors, see commonly assigned International Patent Application PCT/IB03/00059, filed Jan. 10, 2003 (the '059 application), which is herein incorporated by reference in its entirety for all purposes.
The '059 application discloses a particularly potent and selective CDK4 inhibitor, 6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one:

In standard enzyme assays the compound of Formula 1 exhibits IC50 concentrations for CDK4 and CDK2 inhibition (at 25° C.) of 0.011 μM and >5 μM, respectively. For a discussion of standard CDK4 and CDK2 assays for IC50 determinations, see D. W. Fry et al., J. Biol. Chem. (2001) 16617-16623.
Though the compound of Formula 1 is a potent and selective CDK4 inhibitor, its use in pharmaceutical products presents challenges. For example, the free base has poor water solubility (9 μg/mL) and exhibits low bioavailability in animal studies. A di-HCl salt of the compound of Formula 1 appears to exhibit adequate water solubility. However, moisture uptake studies reveal that, even at low relative humidity (10% RH), the di-HCl salt absorbs water in an amount greater than about 2% of its mass, making it unsuitable for use in a solid drug product. A mono-HCl salt of the compound of Formula 1 is marginally hygroscopic, absorbing more than 2% of its mass at a relative humidity above 80%. However, the process for preparing the mono-HCl salt yields partially crystalline drug substance, indicating potential problems with process scale-up. Other salt forms of the compound of Formula 1 are thus needed.